Sequencing Overview

Illumina’s innovative and flexible sequencing system enables a broad array of applications in genomics, transcriptomics, and epigenomics. Libraries are prepared from genomic DNA or RNA, then immobilized on the surface of a flow cell designed to present the DNA in a manner that facilitates access to enzymes while ensuring high stability of surface-bound template and low non-specific binding of fluorescently labeled nucleotides. Solid-phase amplification creates up to 1,000 identical copies of each single template molecule in close proximity with total densities on the order of more than one million single-molecule clusters per square millimeter.

Sequencing by Synthesis

Sequencing by synthesis (SBS) technology uses four fluorescently labeled nucleotides to simultaneously sequence the tens of millions of clusters on the flow cell surface. During each sequencing cycle, a single labeled deoxynucleoside triphosphate (dNTP) is added to the nucleic acid chain. The nucleotide label serves as a terminator for polymerization. After each dNTP incorporation, the fluorescent dye is imaged to identify the base and later enzymatically cleaved to allow incorporation of the next nucleotide. Since all four reversible terminator-bound dNTPs (A, C, T, G) are present as single separate molecules, natural competition minimizes incorporation bias. Base calls are made directly from signal intensity measurements during each cycle, thereby reducing raw error rates. The end result is highly accurate base-by-base sequencing that eliminates sequence-context specific errors, enabling robust base calling across the genome.